Weiss W, Huber G, Engel K H, Pethran A, Dunn M J, Gooley A A, Görg A
Department of Food Technology, Technical University of Munich, Freising-Weihenstephan, Germany.
Electrophoresis. 1997 May;18(5):826-33. doi: 10.1002/elps.1150180529.
Bakers' asthma, an immediate-type allergic response to the inhalation of cereal flours, is an important occupational disease among workers of the baking and milling industries, and the salt-soluble proteins of wheat and rye flour dust are considered the most relevant allergens. In order to identify and characterize the major IgE-binding proteins, the polypeptide composition of the albumin/globulin protein fraction obtained from different cultivars was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and high-resolution two-dimensional polyacrylamide gel electrophoresis with immobilized pH gradients in the first dimension (IPG-Dalt), followed by immunoblotting with sera from asthmatic bakers. Relevant allergens were isolated by micropreparative IPG-Dalt and blotting onto polyvinylidenedifluoride membranes and identified by amino acid composition analysis or N-terminal amino acid sequence analysis. SDS-PAGE, IPG-Dalt, and immunoblotting demonstrated that the sera of the bakers allergic to flour contained IgE antibodies which bound to numerous albumin/globulin polypeptides in the 70, 55, 35, 26-28, and 14-18 kDa areas. More detailed investigations using IPG-Dalt revealed cultivar-specific differences in IgE-binding. It was also demonstrated that the majority of the allergens were not single polypeptide spots, but consisted of up to ten isoforms of similar molecular mass but different isoelectric points. Amino acid composition analysis and N-terminal amino acid sequence analysis, which were performed for nine allergens located in the 14-18, 26-28, and 35 kDa areas, revealed homologies to amylase/protease inhibitors, acyl-CoA oxidase and fructose-bisphosphate-aldolase from wheat, barley, maize, and rice, respectively.
面包师哮喘是一种对吸入谷物面粉产生的速发型过敏反应，是烘焙和磨粉行业工人中的一种重要职业病，小麦和黑麦面粉粉尘中的盐溶性蛋白质被认为是最主要的过敏原。为了鉴定和表征主要的IgE结合蛋白，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分析了从不同品种获得的白蛋白/球蛋白组分的多肽组成，并进行了一维采用固定化pH梯度的高分辨率二维聚丙烯酰胺凝胶电泳（IPG-Dalt），随后用哮喘面包师的血清进行免疫印迹。通过微量制备IPG-Dalt分离相关过敏原，并将其印迹到聚偏二氟乙烯膜上，通过氨基酸组成分析或N端氨基酸序列分析进行鉴定。SDS-PAGE、IPG-Dalt和免疫印迹表明，对面粉过敏的面包师血清中含有IgE抗体，这些抗体与70、55、35、26 - 28和14 - 18 kDa区域的多种白蛋白/球蛋白多肽结合。使用IPG-Dalt进行的更详细研究揭示了IgE结合的品种特异性差异。还证明大多数过敏原不是单一的多肽斑点，而是由多达十种分子量相似但等电点不同的同工型组成。对位于14 - 18、26 - 28和35 kDa区域的九种过敏原进行的氨基酸组成分析和N端氨基酸序列分析分别揭示了与小麦、大麦、玉米和水稻中的淀粉酶/蛋白酶抑制剂、酰基辅酶A氧化酶和果糖二磷酸醛缩酶的同源性。
